Enzymatic hydrolysis of fluorogenic substrates to yield a detectable fluorescent species is known to be useful in a variety of diagnostic applications. For example, a number of researchers have investigated the possibility of using a panel of fluorogenic substrates to determine which enzymes are present in a sample containing an unidentified microorganism and correlating the profile to known profiles to identify the unknown microorganism. Notwithstanding the desirability of using this fluorogenic detection system, widespread use of it has not been adopted for a variety of reasons which make it impracticable.
When laboratories identify organisms from clinical isolates an important goal is rapid identification. Most commercial bacterial identification systems require 18 to 24 hours or longer following isolation of an organism to achieve identification. Some of the current "rapid" systems take 3 to 13 hours. These systems generally rely upon the detection of acidic or basic by-products of sugar or amino acid metabolism produced following a period of organism growth.
One method for identifying specific bacterial species using enzymatic cleavage of substrates is described in U.S. Pat. No. 4,603,108 to Bascomb. The Bascomb patent describes a kit containing tests for 26 constitutive enzymes. In each test the enzyme is determined by its ability to interact with a specific substrate. A test card or other apparatus has a plurality of wells or compartments which separately contain specific substrate solutions for each of the enzyme tests together with other reagents for the tests. In use a bacterial suspension is added to each compartment and a detectable product is developed after a relatively short incubation period. The amount of the corresponding enzyme in each sample is then determined by spectrometric analysis using either colorimetry or fluorimetry.
Another procedure described in the patent uses 7 tests for rapid differentiation of commonly encountered bacterial groups. Bascomb teaches that either the 26 test assay or the 7 test assay gives a unique fingerprint for the species or group of species. A quantitative determination of enzyme activity for each group or species can be used to identify the group or species by comparison to activity profiles of previously identified bacteria. The specific tests described determine activity by detecting absorbance in a flow cell. Discrete sample analysis and continuous flow analysis can be used. The method of the Bascomb patent requires a large biomass and a high fluid volume as well as a relatively long incubation time.
Other scientists have used fluorogenic substrates to identify microorganisms. Westley, J. W. et al, discuss the use of alpha-amino acid B napthylamide substrates for identification of 24 strains of bacteria ("Aminopeptidase Profiles of Various Bacteria"; Appl. Micro., 15:822-825, 1967). Bacteria were suspended in solution and incubated with substrate solutions. The fluorescence of the released B-napthylamine was measured after 4 hours of incubation.
Another paper describing the use of fluorometric analysis to measure enzyme hydrolysis of 19 L-amino acid B-napthylamides is Peterson, E. W. et al., "Rapid Detection of Selected Gram-Negative Bacteria by Aminopeptidase Profiles"; J. Food Sci., 43:1853-1856 (1978). A profile for each culture was obtained in 4 to 6 hours.
A review of the literature pertaining to use of fluorogenic substrates to profile microbial enzyme activity is contained in Godsey, J. H. et al., "Rapid Identification of Enterobacteriaceae with Microbial Enzyme Activity Profiles"; J. Clin. Micro., 13:483-490 (1981). The Godsey group reports use of eighteen fluorogenic substrates in a study of 539 strains of the family Enterobacteriaceae. Hydrolysis rates were monitored for the first 30 minutes in 2 ml of buffer containing substrate at 37.degree. C. All substrates except urea were derivatives of B-methylumbelliferone, B-napthylamine or 7-amino 4-methyl coumarin.
In U.S. Pat. No. 4,591,554, to Koumara et al, fluorescence analysis using umbelli ferone derivatives is described as a method to detect and determine the number of small numbers of microorganisms. In the method an umbelliferone derivative is added to a sample solution and the mixture solution is incubated. Thereafter, insoluble residues (e.g. cells) are removed and fluorescence is read in a conventional detector. The amount of fluorescence is then related to the number of microorganisms. The examples describe experiments where a solution containing the substrate is mixed with a solution containing bacteria. After incubation the pH is adjusted and the mixture is centrifuged to remove insoluble cells. Thereafter the fluorescence of any liberated 4-methylumbelliferone is determined. In some cases coenzymes are used. In other cases the cells are disrupted to increase the amount of liberated enzymes.
Fluorogenic substrates are also known to be useful to assay extracellular enzymes present in living organisms (Snyder, A. et al., "Pattern Recognition Analysis of In-Vivo Enzyme Substrate Fluorescence Velocities in Microorganism Detection and Identification"; App. & Enviro Micro, 51:969-977 (1986)). Reaction times were fifteen minutes or less. Assays were carried out in 2 ml buffer. This work also forms the basis of an International Patent Application entitled "Viable Microorganism Detection by Induced Fluorescence" with the University of Cincinnati as applicant (Int'l Pub. No. WO 86/05206 dated Sep. 12, 1986).
Yet another technique to fingerprint bacteria based on the differences in enzyme content and activity is described in Chou Pong Pau et al, "A Rapid Enzymatic Procedure for Fingenprinting, Bacteria by Using Pattern Recognition of Two-Dimensional Fluorescence Data"; Clin. Chem. 32:987-991 (1986). In that system a mixture of 6 fluorogenic substrates is used, each with a different fluorescent moiety. Fluorescence increases are monitored over a 30-minute period. A Fourier transformation of the fluorescence data is used to produce a two dimensional array which is characteristic of each test organism. This method requires the use of complicated and expensive equipment to perform the measurements and to execute the mathematical transformation.
Use of free fluors in diagnostics is well known. Many free fluors are known to be quenched or enhanced by variations in enviromental conditions such as pH, redox potential or oxygen partial pressure. These fluors are used to detect or monitor the enviromental condition that affects their fluorescence.
Each of the methods described above to identify or quantify the amount of analyte present in a sample by detecting or monitoring enzyme hydrolysis of a fluorogenic substrate requires an aqueous environment. Similarly, when free fluors are used to monitor enviromental changes in a biological test system, an aqueous enviroment is required. Problems arise in designing aqueous test systems using free fluors or fluorogenic substrates and having acceptable shelf lives because free fluors and fluorogenic substrates need dry conditions to best maintain their stability. Thus one design challenge is the problem of providing the free fluor or fluorogenic substrate in a dry state.
When fluorogenic substrates are stored dry, they need to be available to react with the enzyme so that they rapidly reach a steady-state reaction following addition of an aqueous test suspension or solution. This challenge is not easily met because fluorogenic substrates show various solubilities in water. The least water soluble are generally the lipase substrates. This challenge is particularly difficult for those substrates having low aqueous solubility.
One material that has been used as a support for fluorescent materials is cellulose filter paper. Whatman No. 4 paper was used for analysis at 4.degree. K. of pyrene, benzo[a]pyrene, chrysene and solvent-refined-coal by Tuan Vo Dinh, "Fluorescence Line Narrowing Spectrometry of Polycyclic Compounds on Filter Paper Substrates"; Anal. Chem. 58:3135-3139 (1986).
One susceptibility testing product line uses filter paper to store fluorogenic substrates. In Sensititre.TM. Susceptibility panels a product of Radiometer of Copenhagen, Inc., Copenhagen, Denmark) fluorogenic substrates are provided dry on filter paper. In use, the filter paper strips are placed in broth and the fluorogenic substrate is eluted into solution. Thereafter the broth is dispensed into microwells where the solution susceptibility test is performed.
Another problem encountered when characterizing an enzyme profile to identify a microorganism or a pathological state is the problem of inadequate biomass. Desirably in microorganism identification tests, the microorganism to be identified is obtained from an isolated colony from an overnight streak plate prepared from a clinical sample, or directly from a positive blood culture vial. In both of these circumstances the number of microorganisms available is limited. Similarly when a biological sample is tested for endogenous enzyme content, the amount of biological fluid or tissue available for analysis may be limited. Thus the amount of biomass needed to perform the characterization should be minimized. To achieve rapid results, a high biomass concentration is necessary. To satisfy these two criteria, a test system should be miniturized to the extent reasonably possible. Miniaturization to allow use of a small biomass causes another problem. With a given amount of fluorogenic substrate and available enzyme concentration, miniturization of the system reduces the amount of substrate hydrolized per unit time. The total fluorescence change per unit time is also reduced.
Thus a need exists for a miniaturized system which has an acceptable shelf life, requires a small biomass of sample, provides fast equilibration to steady-state kinetics following addition of sample, and yields an enhanced fluorescence signal.